Determining the Architectures and Activities of Polyketide Synthase Modules

模块化聚酮合成酶领域的复兴已经开始。新的工具和模式正在使 对这些酶装配线的架构和活动有更深入的了解，并正在促进我们的 将模块化聚酮合成酶的合成能力应用于开发和 新药的生产。使用最新的“模块”定义，我们的实验室设计了不同的三元模型。 /四-/五酮合酶在体内和体外都有功能。虽然这些短小的装配线是 在自然界中不常见，它们是我们结构和功能研究的理想选择。在具体目标1中，我们建议 当这些装配线合成他们的聚酮产品时，他们突然冻结，并通过 低温电子显微镜。由于这种方法使我们能够捕获高分辨率、动态信息 一个模型三酮合成酶的启动酮合成酶和酰基转移酶，我们将把它应用于 包含其他感兴趣的区域。一个目标是了解酮基还原酶、脱水酶和 烯酰还原酶处理酶是相对于彼此和相邻的 酮合成酶+酰基转移酶双域，以了解酰基载体蛋白结构域如何在这些结构域之间移动 聚酮中间体延伸和加工过程中的酶。因此，我们将至少调查 在我们实验室功能验证的13个工程三/四酮合酶和两个天然合酶 含有不同组的这些加工酶。在具体目标2中，我们建议阐明相互作用 在加工过的聚酮中间体和为它们守护的酮合成酶之间。我们有很强的 底物隧道残基如何与最接近活性硫酯的中间体相互作用的假设 以确保它们被上游加工酶适当地修饰。因此，我们将适当地变异 活性较低的模型合酶以及模型合酶中酮合成酶的把关残基 灭活上游处理酶，并确定其生产率是否如预测的那样提高。 由于我们的数据表明聚酮中间体使酮合成酶二聚体和二聚体刚性 在冷冻电子显微镜研究中，可以很容易地确定酮合成酶+酰基转移酶双域，我们将 还对停滞的合成酶进行电子显微镜检查，以解决聚酮结合的结构 酮合成酶+酰基转移酶二聚体。在具体目标3中，我们建议确定关键的域-域接口。 我们有证据表明加工酶和下游KSS之间的接口驱动有序的自我 合成酶多肽的组装多于观察到的C-端和N-端之间的小界面 对接结构域，寻找具有代表性的络合物结构。我们的目标也是确定酰基载体 在转酰化反应中，蛋白质结构域与酮合成酶对接。如果我们在这些方面取得成功 项目，它将极大地促进合成新的模块化聚酮合成酶的合理工程 分子，最终是新药。

A renaissance in the field of modular polyketide synthases has begun. New tools and paradigms are enabling deeper insights into the architectures and activities of these enzymatic assembly lines and are facilitating our long-term goal of applying the synthetic power of modular polyketide synthases to the development and production of new medicines. Using the updated definition of the “module”, our lab has engineered diverse tri- /tetra-/pentaketide synthases that are functional both in vivo and in vitro. While these short assembly lines are uncommon in nature, they are ideal for our structural and functional studies. In Specific Aim 1 we propose to plunge-freeze these assembly lines as they are synthesizing their polyketide products and investigate them by cryo-electron microscopy. Since this approach has enabled us to capture high-resolution, dynamic information of the priming ketosynthase and acyltransferase of a model triketide synthase, we will apply it to synthases that contain other regions of interest. One objective is to learn how the ketoreductase, dehydratase, and enoylreductase processing enzymes are oriented relative to one another and the neighboring ketosynthase+acyltransferase didomains to understand how acyl carrier protein domains move between these enzymes during the extension and processing of polyketide intermediates. Thus, we will investigate at least thirteen engineered tri-/tetraketide synthases and two natural synthases functionally validated in our lab that contain different sets of these processing enzymes. In Specific Aim 2 we propose to elucidate interactions between processed polyketide intermediates and the ketosynthases that gatekeep for them. We have strong hypotheses for how sets of substrate tunnel residues interact with intermediates closest to the reactive thioester to ensure they are properly modified by upstream processing enzymes. Thus, we will appropriately mutate the gatekeeping residues of ketosynthases in less active model synthases as well as model synthases with inactivated upstream processing enzymes and determine whether their productivities improve as predicted. Since our data indicate that polyketide intermediates rigidify the ketosynthase dimer and dimeric ketosynthase+acyltransferase didomains can be readily identified in cryo-electron microscopy studies, we will also perform electron microscopy on stalled synthases to solve structures of polyketide-bound ketosynthase+acyltransferase dimers. In Specific Aim 3 we propose to determine key domain-domain interfaces. We have evidence that interfaces between processing enzymes and downstream KSs drive the ordered self- assembly of synthase polypeptides more than the small interface observed between the C- and N-terminal docking domains and seek structures of representative complexes. We also aim to determine how acyl carrier protein domains dock with ketosynthases during the transacylation reaction. If we are successful in these projects, it will greatly inform the rational engineering of modular polyketide synthases that synthesize new molecules and, ultimately, new medicines.